Role of Intracellular Oxidant Release in Oxidised Low Lipoprotein - Induced U937 Cell Death

Abstract

Atherosclerosis is a complex inflammation condition involving the accumulation of lipid-filled macrophages within the artery wall. Progression of the initial fatty streak to an advanced atherosclerotic plaque is characterized by the development of a necrotic core region containing cholesterol and dead cells. The oxidation of low-density lipoprotein (LDL) to oxidized LDL (oxLDL) and its subsequent uptake by macrophages to form foam cells are the key process in plaque formation. OxLDL is found within atherosclerotic plaque, and it is cytotoxic to a range of cells including macrophages through the generation of reactive oxygen species (ROS) and induction of oxidative stress.

The aim of this study was to examine the cytotoxic effects of oxLDL to U937 human monocyte-like cells. OxLDL caused a rapid concentration-dependent cell viability loss in U937 cells within 6 hours. The progression of oxLDL-induced cell death was found to be strongly correlated with the intracellular ROS production and intracellular glutathione (GSH) loss. OxLDL also caused a rapid loss of intracellular aconitase activity, indicating the impairment of the cellular metabolic function. The cytosolic calcium ion (Ca²⁺) level was also elevated by oxLDL, which could be from both intra- and extra-cellular sources.

OxLDL also activated plasma membrane superoxide generation complex NADPH oxidase (NOX), and the progression of oxLDL-induced NOX activation was correlated with oxLDL-mediated ROS production, suggesting NOX is the major source of ROS. Further investigations using NOX inhibitors apocynin or diphenyleneiodonium (DPI) found that inhibition of NOX prevented oxLDL-induced cell viability loss, ROS production, GSH loss and aconitase activity decrease. The cytosolic Ca²⁺ elevation caused by oxLDL was also suppressed slightly by inhibiting NOX activity. These results clearly show that NOX is the major site of oxidative stress upon oxLDL activation, contributing to the oxLDL-induced cell death.

This study also examined the protective effect of 7,8-dihydroneopterin (7,8-NP) on oxLDL-induced oxidative stress. 7,8-NP dramatically protected cells from oxLDL-induced cell viability loss, ROS generation and aconitase activity loss. 7,8-NP also inhibited oxLDL-induced cytosolic Ca²⁺ influx particularly after 3 hours. 7,8-NP did not inhibited mitochondrial aconitase activity decrease caused by oxLDL, nor inhibited mitochondrial ROS production. This indicates the protective effect of 7,8-NP against oxLDL damage could primarily in cytoplasm. The failure of 7,8-NP protection from oxLDL activating NOX suggests that the protection of 7,8-NP against oxLDL-induced oxidative stress was not due to the inhibition of NOX activation, but by radical scavenging activity of the NOX products.